An improved isolation procedure has been developed for bovine liver mitochondrial branched chain ketoacid dehydrogenase complex. With this procedure the multienzyme complex is obtained in high yield (greater than 30%) and with a high specific activity (2-6 micromoles/min/mg protein). The complex is inhibited by its reaction products, NADH and the branched chain acyl-CoA thiol esters. These compounds may serve a regulatory function in controlling this rate-limiting reaction of leucine, isoleucine, and valine catabolism in vivo. Two other inhibitors of the complex which have physiologic significance are clofibric acid, and antihyperlipidemic drug, and phenylpyruvic acid, a minor catabolite of tissues. Clofibric acid causes 50% inhibition at 0.24 mM while phenylpyruvate reduces activity of the complex by 50% at 0.4 mM. These concentrations are found in blood and tissues of patients being treated with clofibric acid while the high level of phenylpyruvate can be seen physiologically in untreated phenylketonuria. Both compounds form abortive enzyme-substrate-inhibitor complexes. Clofibric acid binds preferentially to the enzyme-substrate complex while phenylpyruvate prefers binding to free enzyme. These compounds can be chemically transformed into photoreactive molecules which will be used in further characterization studies. Both compounds also work as inhibitors in animals and cultured cells. Therefore, they can be used to create model systems for studying the consequences of impaired branched chain ketoacid dehydrogenase.